Genome instability quantified in constitutive tissues as well as putatively stable tumor tissue in hereditary non -polyposis colon cancer patients by small pool PCR

Microsatellite instability (MSI) is a hallmark of the mutator phenotype associated with Hereditary Non-Polyposis Colon Cancer (HNPCC). The MSI-High (MSI-H) HNPCC population has been well characterized, but the microsatellite low and stable (MSI-L/MSS) HNPCC population is much less understood. We hypothesize there are significant levels of MSI in HNPCC DNA classified as MSI-L/MSS, but no single variant allele makes up a sufficient population in the tumor DNA to be detected by standard analysis. Finding variants would suggest there is a mutator phenotype for the MSI-L/MSS HNPCC population that is distinct from the MSI-H HNPCC populations. This study quantified and compared MSI in HNPCC patients previously shown to be MSI-H, MSI-L/MSS and an MSI-H older, sporadic colorectal cancer patient. Small-pool Polymerase Chain Reactions (SP-PCRs) were conducted where the DNAs from each sample and controls are diluted into multiple pools, each containing approximately single genome equivalents. At least 100 alleles/sample were studied at six microsatellite loci. Mutant fragments were identified, quantified, and compared using Poisson statistics. Most of the variants were small deletions or insertions, with more mutants being deletions, as has been previously described in yeast and transgenic mice. SP-PCR, where most of the pools contained only 3 or less fragments, enabled identification of variants too infrequent to be detected by large pool PCR. Mutant fragments in positive control MSI-H tumor samples ranged from 0.26 to 0.68 in at least 4 of the 6 loci tested and were consistent with their MSI-H status. In the so called MSS tumors and constitutive tissues (normal colon tissue, and PBLs) of all the HNPCC patients, low, but significant levels of MSI were seen in at least two of the loci studied. This phenomenon was not seen in the sporadic MSI constitutive tissues nor the normal controls and suggests haploinsufficiency, gain-of-function, or a dominant/negative basis of the instability in HNPCC patients carrying germline mutations for tumor suppressor genes. A different frequency and spectrum of mutant fragments suggests a different genetic basis (other than a major mutation in MLH1 or MSH2) for disease in MSI-L and MSS HNPCC patients. ^

Early stages of experimental colon carcinogenesis inhibit mast cell-dependent mucosal immune function of the distal colon

Inhibition of local host immune reactions is one mechanism contributing to tumor progression. To determine if alterations in local immune functioning occur during colon carcinogenesis, a model mucosal immune response, type I hypersensitivity against the intestinal parasite Trichinella spiralis, was first characterized in normal mice and then examined during experimental colon carcinogenesis. Segments of sensitized colon mounted in Ussing chambers and challenged with T. spiralis-derived antigen resulted in a rise in short-circuit current ($\rm\Delta I\sb{sc}$) that was antigen-specific and inhibited by furosemide, implicating epithelial Cl$\sp-$ secretion as the ionic mechanism. The immune-regulated Cl$\sp-$ secretion by colonic epithelial cells required the presence of mast cells with surface IgE. Inhibition of potential anaphylactic mediators with various pharmacological agents in vitro implicated prostaglandins and leukotrienes as the principal mediators of the antigen-induced $\rm\Delta I\sb{sc}$, with 5-hydroxytryptamine also playing a role. Distal colon from immune mice fed an aspirin-containing diet (800 mg/kg powdered diet) ad libitum for 6 wk had a decreased response to antigen, confirming the major role of prostaglandins in generating the colonic I$\sb{\rm sc}$. To determine the effects of early stages of colon carcinogenesis on this mucosal immune response, mice were immunized with T. spiralis 1 day after or 8 wk prior to the first of 6 weekly injections of the procarcinogen 1,2-dimethylhydrazine (DMH). Responsiveness to antigenic challenge was suppressed in the distal colon 4-6 wk after the final injection of DMH. One injection of DMH was not sufficient to inhibit antigen responsiveness. The colonic epithelium remained sensitive to direct stimulation by exogenous Cl$\sp-$ secretagogues. Decreased antigen-induced $\rm\Delta I\sb{sc}$ in the distal colon was not due to systemic immune suppression by DMH, as the proximal colon and jejunum maintained responsiveness to antigen. Also, rejection of a secondary T. spiralis infection from the small intestine was not altered. Tumors eventually developed 25-30 wk after the final injection of DMH only in the distal portions of the colon. These results suggest that early stages of DMH-induced colon carcinogenesis manipulate the microenvironment such that mucosal immune function, as measured by immune-regulated Cl$\sp-$ secretion, is suppressed in the distal colon, but not in other regions of the gut. Future elucidation of the mechanisms by which this localized inhibition of immune-mediated ion transport occurs may provide possible clues to the microenvironmental changes necessary for tumor progression in the distal colon. ^